at-glucosidase 11-deficient Cells Use Endo at-mannosidase as a Bypass Route for N-Linked Oligosaccharide Processing*

THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 266. No. 6, Issue of February 25, pp. 3571-3578, 1991 Printed in U.S.A. at-glucosidase 11-deficient Cells Use Endo at-mannosidase as a Bypass Route for N-Linked Oligosaccharide Processing* (Received for publication, September 20, 1990) Karen Fujimoto and Rosalind Kornfeld From the Department of Internal Medicine, Division of Hematology-Oncology and the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 The kinetics of N-linked oligosaccharide processing may be of the high mannose, hybrid, or complex type dependand the structures of the processing intermediates have ing on the particular glycoprotein and the specific glycosylabeen examined in normal parental BW5147 mouse tion site. The pathway of oligosaccharide processing is fairly lymphoma cells and the a-glucosidase 11-deficient well understood, and many of the enzymes have been studied PHAR2.7 mutant cells. The mutant cells accumulated and purified. A key requisite to the formation of complex glucosylated intermediates but were able to degluco- oligosaccharides is the early removal of the 3 glucose residues sylate and process about 40% of their oligosaccharides which block the al,3-linked arm of the high mannose precurto complex-type. This processing was not due to residsor from further processing. In 1982, Reitman et al. (3) deual a-glucosidase I1 activity since the a-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butylscribed a mutant mouse lymphoma cell line (PHAR2.7), se- DNJ did not prevent it. Parent cells also showed a- lected for resistance to the lectin leukoagglutinating phytoglucosidase 11-independent processing in the presence hemagglutinin, which was severely deficient in a-glucosidase of DNJ and N-butyl-DNJ. Membrane preparations I1 activity. The asparagine-linked oligosaccharides of the mufrom both parent and mutant cells had endo a-manno- tant cells were greatly enriched in GlcpMan8-gGlcNAcZ strucsidase activity, that is, split GlcI,zMan9GlcNAc to tures indicating that a-glucosidase I1 activity was responsible Glcl,zMan plus MansGlcNAc, indicating that this was for removal of both inner glucose residues. These mutant cells a candidate for an alternate route to complex oligosac- made fewer complex oligosaccharides, which accounted for charide formation in the mutant cells. A balance study the decreased binding of leukoagglutinating phytohemaggluin which the cellular glycoproteins, intracellular water tinin to the cell surface and hence their lower sensitivity to soluble saccharides, and saccharides secreted into the leukoagglutinating phytohemagglutinin toxicity. However, medium were isolated and analyzed from [2-3H]man- despite their profound enzyme deficiency, the mutant cells nose-labeled mutant cells showed that the cells formed still processed a significant proportion of their oligosacchathe di- and trisaccharides GlqMan andglczman in rides to complex-type structures. We undertook the present amounts equivalent to the deglucosylated oligosacchastudy to explore the possible explanations for this paradox. rides found in the cellular glycoproteins. This result One possibility was that the in vitro assays of a-glucosidase shows unequivocally that the a-glucosidase 11-deficient mutant cells use endo a-mannosidase as a bypass route I1 activity do not reflect its true in vivo activity in the mutant for N-linked oligosaccharide processing. whether due to instability of the enzyme or altered ph optimum or K,,, for substrate. A related possibility was that normal cells contain a huge excess of a-glucosidase I1 and the 0.3-3% of activity in the mutant isufficient to deglucosylate 50% of the oligosaccharides. A different possibility was that the mu- Asparagine-linked oligosaccharides are attached to nascent tant cells have a pathway or mechanism to circumvent the a- polypeptides in the rough endoplasmic reticulum (ER), and the initial glycosylated product has the structure Glcal-2 glucosidase I1 deficiency. For example, Romero and Herscov- Glcal-3 Glcal-3 MangGlcNAcz (see Refs. 1 and 2 for reics (4) found that F9 teratocarcinoma cells can circumvent an views). This structure is sequentially modified in the ER by a-glucosidase I1 inhibitor block by transferring non-glucothe action of a-glucosidase I which removes the outer a-1,2- sylated lipid-linked oligosaccharide precursor to asparagine linked glucose residue and a-glucosidase I1 which removes the inner two al,3-linked glucose residues. As the newly synthe- sized glycoproteins move through the ER and Golgi apparatus, their deglucosylated oligosaccharides are exposed to a variety of a-mannosidases and glycosyl transferases which complete the oligosaccharide processing. The mature oligosaccharide * This work was supported by Grant CA08759 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: ER, endoplasmic reticulum; DNJ, deoxynojirimycin; bu-dnj, N-butyl-deoxynojirimycin; MES, 4-morpholineethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC, high performance liquid chromatog- raphy; endo H, endo-8-n-acetylglucosaminidase H; ConA, concanavalin A. 3571 residues. However, the most intriguing possibility for an a- glucosidase 11-independent pathway was the enzyme endo a- mannosidase, recently discovered by Lubas and Spiro (5) in rat liver Golgi, which splits GlclMangGlcNAcz to give the disaccharide GlclMan plus Man8GlcNAc2. The endo a-mannosidase was also shown to act on GlcaMangGlcNAc2 and GlczMangGlcNAcz to release the tetrasaccharide and trisaccharide Glc,Man and GlczMan (6). In this study we demonstrate that the PHAR2.7 mutant cells employ the endo a- mannosidase as a bypass route to process their oligosaccharides to complex-type structures. EXPERIMENTAL PROCEDURES Materials-Materials were obtained from the following sources: D- [2- H]mannose (15 Ci/mmol), American Radiolabeled Chemicals, Inc., St. Louis, MO; ATP, swainsonine, methyl-a-d-mannopyranoside, Sephadex G-25-80, and Amberlite MB-3, Sigma; Scintiverse I,

3572 Oligosaccharide Processing in a-glc II-deficient Cells Fisher; MES and HEPES, Behring Diagnostics; ConA-Sepharose, oligosaccharide peaks were treated with a-mannosidase (0.70 unit in Pharmacia LKB Biotechnology Inc.; HPLC Micro Pak AX-5 col- 50 pl of 50 mm citrate buffer, ph 4.5, at 37 "C for 48 h) which will umns, Varian; methyl a-d-glucopyranoside, Aldrich; l-deoxynojiri- degrade non-glucosylated oligosaccharides to free mannose plus mycin (DNJ) and deoxymannojirimycin, Genzyme; and N-butyl- ManGlcNAc and glucosylated oligosaccharides to free mannose plus deoxynojirimycin (bu-dnj) was a kind gift from Drs. D. Tiemeier either GlclMan,GlcNAc, GlczMan4GlcNAc, or Glc3Man4GlcNAc. The and R. Mueller, Searle, St. Louis, MO. Fetal calf serum was from a-mannosidase reaction products were separated either by HPLC or Armour Pharmaceutical Co., and a-minimal essential medium and by paper chromatography in solvent A, both of which resolve the other media were supplied by the Washington University Tissue three different glucosylated products. To ensure that the a-mannos- Culture Support Center. idase digestions had gone to completion, standard oligosaccharides Enzymes-Pronase, grade B, was from Behring Diagnostics, yeast Man&lcNAc and GlclMan9GlcNAc were incubated and worked up hexokinase was from Boehringer Mannheim, and endo 6-N-acetyl- under the same conditions. glucosaminidase H (endo H) was from Miles. Jack bean a-mannosi- The distribution of radioactive oligosaccharides in ConA-Sephadase was prepared as described by Li and Li (7) and baker's yeast a- rose fractions was converted to mol % of total oligosaccharide by glucosidase (Type I) was purchased from Sigma. compensating for the number of radioactive sugar residues in each Cells-The selection of the mutant cell line (PHAR2.7) from the oligosaccharide molecule. For example, 100 cpm of MangGlcNAc parent BW 5147 mouse lymphoma cell line has been described (8). equals 100/9 or 11.1 parts of MangGlcNAc, 100 cpm of Man6GlcNAc Cells were grown in suspension without agitation in a-minimal essen- equals 100/6 or 16.7 parts of Man6GlcNAc, and 100 cpm of the tial medium supplemented with heat-inactivated fetal calf serum complex oligosaccharides in ConA-Sepharose peaks I and 11, which (lo%), penicillin (50 units/ml), and streptomycin (50 pglml), at 37 'C in a humidified atmosphere of 95% air, 5% CO,. contain 3 residues of mannose and 0.5 residue of fucose/molecule, equals 100/3.5 or 28.6 parts of complex species. When the total parts Labeling of Cells and Preparation of Glycopeptides-Cells were of all species are summed and set to loo%, it is then possible to harvested, washed with Earle's minimal essential medium minus express the amount of each species as mol % of the total. In the glucose and bicarbonate but buffered with 20 mm HEPES, ph 7.5, example just given, the total parts are 56.4, and Man9GlcNAc repreand supplemented with 2 mm glutamine, and resuspended in the same sents 19.7 mol %, Man&lcNAc 29.6 mol %, and complex species 50.7 medium and incubated for 10 min at 37 "C. For each time point of mol % of the total oligosaccharide molecules. the chase, 1 X lo7 cells in 1.0 ml of the above medium were pulse Isolation and Characterization of Secreted Trisaccharide and Disaclabeled with 100 FCi of [2-3H]mannose for 15 min at 37 "C, and the charide-cells (1 X IO7) of both mutant and parent were labeled for chase was initiated by the addition of 9 ml of prewarmed chase 30 min with 500 pci of [2-3H]mannose in 1 ml exactly as described medium, complete a-minimal essential medium with penicillin and above. Then the samples were chilled on ice and washed first with 9 streptomycin, 20 mm HEPS, ph 7.5, 10 mm mannose, and 10% fetal ml of ice-cold glucose-free medium, then with 5 ml, and the cells calf serum. The tubes were incubated on a roller apparatus at 37 "C for the appropriate time of chase. For zero chase the cells were diluted gently resuspended and transferred to a clean tube for the third wash with 5 ml medium. The cells were then resuspended in prewarmed with 9 ml of ice-cold chase medium and kept on ice. To stop the chase chase medium, which contained low glucose (100 p ~ and ) no mannose the samples were chilled and centrifuged at 4 "C to pellet the cells. The cell pellets were washed twice with ice-cold phosphate-buffered saline and stored frozen. After thawing, the cell pellets were extracted as follows: three times with chloroform/methanol(2:1) by sonication first in 3 ml with a 15-min room temperature incubation followed by two extractions with 2 ml and centrifugations to pellet the residue; three times with water by gently drying the residues with a stream of N, and extracting sequentially with 3,2 and 2 ml of HzO; three times with 3,2 and 2 ml of chloroform/methanol/h,o (1010:3) after first resuspending the water extracted pellet in 500 p1of methanol and gently drying with a stream of N,. The extracted cell pellets were then digested with Pronase (1 mg/ml) in 1 ml of 100 mm Tris, ph 8, 20 mm CaC1, at 57 "C for 48 h to generate the cellular glycopeptides. Fractionation of Glycopeptides on Cod-Sepharose-The Pronase digests were stopped by boiling, and the glycopeptides were desalted on columns of Sephadex G-25 prior to fractionation on columns of ConA-Sepharose (3 ml bed) equilibrated in 10 mm Tris-HC1, ph 8, 150 mm NaCl, 1 mm CaCl,, and 1 mmmgc1, (buffer A) at room temperature. Each column was eluted sequentially with 10,3-ml fractions of buffer A, 15,3-ml fractions of buffer A containing 10 mm a- methylglucoside, and finally with 10,3-ml fractions of buffer A containing 0.1 M a-methylmannoside, prewarmed to 57 "C. The fractions were well mixed, an aliquot counted for radioactivity (0.4 ml of water and sample plus 4 ml of Scintiverse I), and then pooled as peaks I, 11,111, and IV as shown in Fig. 1. Preparation and Analysis of High Mannose Oligosaccharides-The ConA-Sepharose peaks 111 and IV were each desalted on columns of but the usual 10% fetal calf serum, and incubated at 37 "C on a roller apparatus for 3 h. After the chase period, cells were pelleted, washed, and worked-up as described above, and the medium from each sample was lyophilized. The media were reconstituted in 1 ml of water, centrifuged to remove some insoluble material and passed over a Sephadex G-25 column which resolved a small turbid peak of radio- activity in the void volume from a large included peak of radioactivity eluting in the area of standard maltotriose and earlier than the phenol red derived from the medium. The peak fractions were pooled, reduced in volume, boiled for 3 min, and then incubated at ph 8 with hexokinase (2.8 units) in the presence of 5 mm ATP and 10 mm MgC1, in a final volume of 1 ml at 37 "C for 17 h under a toluene atmosphere to convert the residual [2-3H]mannose and glucose from the medium to their respective phosphorylated derivatives. The reaction mixtures were boiled 3 min, and the samples were deionized by passage over columns of Amberlite MB-3 that were washed exten- sively with water and the neutral saccharides collected, taken to dryness, and resuspended in a small volume for paper chromatography in solvent B. The hexokinase/amberlite procedure removed 80% of the radioactivity in the samples, but the major peak of radioactivity on the first paper chromatogram was still mannose so the area corresponding very broadly to the di- and trisaccharide region was pooled and subjected to a second paper chromatographic separation in solvent B. The trisaccharide and disaccharide peaks from the second paper chromatographic separation of the mutant secretions (Fig. 5) were pooled conservatively and saccharides subjected to digestion in 50 p1 of reaction volumes with 1) no enzyme in 0.1 M Sephadex G-25, concentrated to dryness, and resuspended in 50 pl of 0.1 M citrate buffer, ph 5.5, to which 2 pl (0.002 unit) of endoglycosodium acetate, ph 6,2) 2 units of yeast a-glucosidase in 0.1 M sodium acetate, ph 6, or 3) 0.7 units of Jack bean a-mannosidase plus 0.8 sidase H was added and incubated under a toluene atmosphere at mm DNJ in 25 mm citrate buffer, ph 4.5, for 24 h at 37 "C under a 37 'C for 48 h. The reactions were stopped by boiling and the released toluene atmosphere. The reaction mixtures were boiled, deionized on high mannose oligosaccharides recovered by passage of the reaction Amberlite MB-3, and the products separated by paper chromatogramixtures over columns of the mixed bed ion-exchange resin Amberlite phy in solvent B. Control incubations of an authentic [2-3H]mannose- MB-3 which were extensively washed with water. The oligosaccharide samples were dried, reconstituted in acetonitrile/h,o (62:38), and size fractionated by HPLC on a Varian 5000 liquid chromatograph labeled Man6GlcNAc under condition 2 showed no release of mannose and under condition 3 showed complete degradation to ManGlcNAc plus free mannose. using a Varian Micro-Pak Ax-5 column by the method of Mellis and Paper Chromatography-Descending paper chromatography was Baenziger (9) as previously described (10). Oligosaccharides ranging performed using Whatman 1 paper with the following solvent sysfrom ManSGlcNAc to MansGlcNAc are well separated on this column tems: solvent A, ethyl acetatelpyridinelacetic acidlwater (5:51:3) and GlclMan9GlcNAc is reasonably well separated from MangGlcNAc and solvent B, butanol/pyridine/water (6:4:3). Sugar standards were and Glc3MangGlcNAc, but GlclMan9GlcNAc is not resolved from visualized by the alkaline silver nitrate technique (11). Radiolabeled GlclMangGlcNAc and likewise Glc,ManeGlcNAc/GlcZManaGlcNAc sugars and oligosaccharides were detected by cutting the paper into and GlclMan7GlcNAc/Glc2Man7GlcNAc overlap each other. To de- 1-cm strips, and for analytical chromatograms the entire strip termine which oligosaccharides were glucosylated and whether with (snipped into pieces) was put into a vial with 0.4 ml of water to which 1, 2, or 3 glucose residues, aliquots of the pooled HPLC-purified 4 ml of Scintiverse I was added for counting in a scintillation spec-

trometer. For preparative chromatograms each strip was snipped into a vial and eluted with 1 mlof water fromwhich an aliquot was counted. Assay of Endo a-mannosidase Activity in Cell Membranes-Cells (1.5 X 10')were harvested, washed with cold phosphate-buffered saline, resuspended in 1 ml of 0.1 M sodium MES, ph 7, and disrupted by sonication on ice for three 10-s bursts at setting 100 on a Bronwill Biosonik IV. The sonicates were centifuged at 133,000 X g for 1 h, and the membrane pellet was resuspended in 1 ml of 0.1 M MES, ph 7, by sonication as before. The assay for endo a-mannosidase was as described by Lubas and Spiro (5, 6) for rat liver Golgi membranes except that we included the a-mannosidase inhibitors deoxymannojirimycin (1 mm) and swainsonine (10 pm) in addition to the EDTA (10 mm) and the a-glucosidase inhibitor DNJ (1 mm) in 0.1 M NaMES, ph 7, 0.2% Triton X-100 for the 45-min preincubation of the membranes at 4 "C. The 50-p1 reaction mixtures, of the same composition as the preincubation mix, contained membranes (75-400 of pg protein) and 8,000-10,000 cpm [2-3H]mannose-labeled GlclMangGlcNAc, (HPLC purified from parent BW 5147 cells) or GlczMangGlcNAc, Glc2Man8GlcNAc, or GlczMan7GlcNAc (HPLC purified from mutant PHAR2.7 cells). Following incubation at 37 "C for 1-3 h, the assays were terminated by methanol precipitation as described (5, 6), and the products were separated by paper chromatography in Solvent A. Protein Determination-Protein concentrations were measured by the method by Lowry et al. (12) using bovine serum albumin as the standard. Oligosaccharide Processing in 11-deficient a-glc Cells 3573 RESULTS AND DISCUSSION charides in fractions I and I1 have, on average, 3.5 radioactive Kinetics of Processing-To answer the question, what is the sugars/chain (3 mannose, 0.5 fucose, data not shown), whereas rate and extent of complex oligosaccharide synthesis in the the high mannose oligosaccharides in fractions I11 and IV a-glucosidase 11-deficient mutant cells, we compared the ki- contain from 5 to 9 radioactive mannose residues/chain. Fracnetics of oligosaccharide processing in the parent and mutant tion I11 contained most, but not all, of the Man5GlcNAc in cells. The cells were labeled for 15 min with [2-3H]mannose, parent cells as well as lesser amounts of Man&lcNAc which chased for 0, 1, 3, or 6 h, and the total cellular glycoproteins fractionated primarily into fraction IV. In mutant cells fracwere digested to glycopeptides with Pronase. The glycopep- tion I11 contained Man6GlcNAc, less Man&lcNAc, and tides were then fractionated on columns of ConA-Sepharose, most of the G1_2Man7GlcNAc. In Table I the high mannose as shown in Fig. 1, to separate tri- and tetraantennary complex species of each type derived from fractions I11 and IV are glycopeptides which do not bind (fraction I) from biantennary combined. complex glycopeptides that elute early with 10 mm a-methylglucoside (fraction 11) and hybrid/small high mannose gly- 000 PARENT CELLS ameglc amemon 1 ' Ohr R 1 400 300 200 300 200 IO0 =idlll 200 IO 20 30 FRA 300 MUTANT CELLS omegk amemon t lr 6hr 1 0 0 1 ~ CTlON IO 20 30 FIG. 1. Separation on ConA-Sepharose of the [2-3H]mannose-labeled glycopeptides formed after various chase periods in parent and mutant cells. Cells were pulse labeled for 15 min with [2-3H]mannose and chased for 0, 1, 3, or 6 h as described under "Experimental Procedures." The Pronase-digested glycopeptides were fractionated on ConA-Sepharose columns, eluting first with 10 mm a-methylglucoside (a-meglc) and then with 100 mm a-methylmannoside (a-meman). Aliquots of each fraction were counted and the fractions were pooled as peaks I (1-5), II (11-16), III (18-25), and IV (26-33). copeptides that elute late with 10 mm a-methylglucoside (fraction 111) and larger high mannose glycopeptides that elute with 100 mm a-methyl mannoside (fraction IV). The elution profiles in Fig. 1 show that both the rate and extent of formation of complex-type glycopeptides in peaks I and I1 are lower in the mutant cells. To examine in more detail the processing intermediates, fractions I11 and IV were treated with endo H to release the high mannose-type oligosaccharides which were size fractionated by HPLC. The isolated oligosaccharides of each size were then treated with a-mannosidase to determine whether they were glucosylated. Nonglucosylated oligosaccharides are converted to free mannose and ManGlcNAc by this treatment, whereas glucosylated oligosaccharides are blocked to a-mannosidase digestion on the glucosylated branch and produce free mannose and either GlclMan4GlcNAc, GlcpMan4GlcNAc, or GlcsMan4GlcNAc depending on their extent of glucosylation. The results of this analysis are summarized in Table I where the data are expressed as the mol % each oligosaccharide species contributes to the total oligosaccharide chains. Because this method normalizes for the number of radioactive sugar residues/oligosaccharide chain, the complex oligosaccharides from fractions I and I1 represent a larger mol % of the total than they represent as % cpm of total cpm. This is because the complex oligosac- Several notable differences can be seen between the oligosaccharides in the normal and mutant cells. The mutant cells form less, but still substantial amounts of complex oligosaccharides. The parent cells contain less glucosylated oligosaccharides (Glc3MangGlcNAc and GlclMangGlcNAc) which decline rapidly over time compared to the mutant cells which contain large amounts of glucosylated oligosaccharides, primarily having 2 glucose residues, that persist throughout the 6-h chase. The fact that the mutant cells do form GlclMangGlcNAc, GlclMan8GlcNAc, and GlclMan7GlcNAc but not MangGlcNAc or Man8GlcNAc suggests that the residual a-glucosidase I1 activity in the mutant cells does convert some GlcnMangGlcNAc to GlclMangGlcNAc, but does not effectively remove the innermost glucose residue from GlclMangGlcNAc. In another experiment, not shown, both cellular and secreted glycoproteins were analyzed after a 15- min pulse label and 6-h chase. The cells secreted very little labeled glycoprotein, which amounted to 3.9% as much radioactivity as that in intracellular glycoproteins for parent cells and 4.5% for mutant cells. The high mannose glycopeptides from mutant cell-secreted glycoproteins also contained glucosylated species. This experiment indicated that the analysis of cellular glycoproteins includes at least 95% of glycoproteins labeled during the pulse. To better evaluate the kinetics of oligosaccharide processing, the data in Table I, including a summation of all gluco- sylated oligosaccharides (i.e. G1-3M7-9) and a summation of all species containing 9 mannose residues (ie. Go-3Mang), have been plotted on a semi-log plot in Fig. 2. This plot shows that in both parent and mutant cells the precursor oligosaccharide Glc3MangGlcNAc disappears with a tl,* of less than 30

3574 Oligosaccharide Processing in 11-deficient a-glc TABLE I Summary of oligosaccharide distribution in cellular glycopeptides at different times of chase M5 to Mg = Man&lcNAc to MangGlcNAc; G1M7 to G1Mg = Glc,Man7GlcNAc to GlclMan9GlcNAc; G2M7 to G3M9 = GlcpMan7GlcNAc to Glc3MangGlcNAc. Parent cells Mutant cells Oligosaccharide species 0 1 3 6h 0 1 3 6h mol % of oligosaccharide Complex 11.6 37.2 52.8 60.8 14.9 23.1 36.1 41.9 Mh 3.8 3.6 5.1 7.2-1.3 0.6 0.8 M6 1.4 2.2 4.4 4.0-2.4 2.4 2.7 M7 5.4 2.4 3.4 5.6-2.5 3.6 3.7 MM 4.2 8.4 8.4 8.0 - - - - M9 32.7 27.8 16.1 11.8 - - - - GIM, - - - - 0.5 1.5 2.5 2.2 GMM - - - - 0.9 1.4 1.4 1.7 41.1 17.4 7.6 2.7 8.6 4.4 1.2 0.9 GN, - - - - 3.0 8.4 14.3 12.2 GMM - - - - 7.8 12.2 12.6 15.7 GJ% - - - - 45.0 39.0 23.7 16.5 G&% 2.8 - - - 19.3 3.8 1.6 1.6 GO-,Mg (all M9 species) 76.6 45.2 23.7 14.5 72.9 47.2 26.5 19.0 G1-3M7-9 (all glucosylated oligos) - - - - 85.1 70.7 57.3 50.8 Species not detected. Cells MUTANT PARENT CELLS I \ CELLS I, we summed all the oligosaccharide species containing 9 mannose residues and found that they disappeared at comparable rates in both cell types, ts 77 min in parent and tlh 90 min in mutant cells. This result indicates that a-mannosidase trimming occurs later than a-glucosidase action, as expected, and that it occurs on the glucosylated oligosaccharides in the mutant cells. Finally, the rate of formation of complex oligosaccharides was plotted, and although by 6 h the mutant cells had formed two-thirds as much complex oligosaccharide as the parent cells, the initial rate was only 8.2 mol %/h compared to 25.6 mol %/h for the parent cells. This sluggish rate of formation parallels the sluggish rate of disappearance of glucosylated oligosaccharides in the mutant cells. Effects of a-glucosidase Inhibitors-The kinetics of processing seen in the pulse-chase study suggested that residual a-glucosidase I1 activity in the mutant cells probably did not account for all the deglucosylation that occurred, but some other mechanism, acting later in the endo-membrane secretory pathway, was responsible. We reasoned that if residual cy-glucosidase I1 activity in the mutant was responsible for oligosaccharide processing, then addition of a potent a-glu- HOURS OF CHASE FIG. 2. Rates of oligosaccharide processing in parent and cosidase I1 inhibitor should prevent the formation of complex oligosaccharides in both mutant and parent cells. Accordingly, mutant cells. The mol % of each oligosaccharide species in parent a 15-min pulse, 6-h chase was performed in the presence of and mutant cells, as tabulated in Table I, is plotted in semi-log either no inhibitor, 4 mm DNJ which primarily inhibits a- coordinates as a function of chase time and the th (half-time of glucosidase I1 (13) or 2 mm bu-dnj which inhibits both a- disappearance) calculated from the initial rate of disappearance. G,Mans is Glc3MangGlcNAc, GIMans is GlclMangGlcNAc, G,,Mans glucosidase I and 11. The elution profiles of the cellular is Glco.3Man9GlcNAc or the summation of all oligosaccharides with glycopeptides on ConA-Sepharose are shown in Fig. 3 and 9 mannose residues, and G1.3Man7_9 is Gl~~.~Man~-~GlcNAc or the reveal that neither inhibitor had a significant effect on the summation of all glucosylated oligosaccharides in the mutant cells. formation of complex oligosaccharides in mutant cells, whereas both inhibitors had an inhibitory effect on complex min consistent with the rapid early action of a-glucosidase I oligosaccharide formation by the parent cells. In fact, the in the ER. In the parent cells the intermediate GlclMang- inhibited parent cell ConA-Sepharose profile is almost iden- GlcNAc disappears somewhat more slowly with a tlh of 48 min tical to the noninhibited mutant cell profile, leading to the consistent with somewhat slower, later action of a-glucosidase conclusion that both cell types contain an a-glucosidase II- I1 in the ER. Since the mutant cells do not show conversion independent pathway for oligosaccharide processing. The exof GlclMangGlcNAc to MangGlcNAc we summed all the glu- istence in the parent cells of the adjunct pathway became cosylated species and plotted them look to for the overall rate apparent only when normal processing was blocked. of deglucosylation which gave a tc of 4 h. In an effort to Since it was of interest to compare the oligosaccharide compare the rates of a-mannosidase trimming which could be intermediates in the processing pathways under both normal caused by either ER a-mannosidases or Golgi a-mannosidase and inhibited conditions, fractions I11 and IV were treated

ol PARENT CELLS Control +DNJ 500[ Oligosaccharide Processing in a-glc 11-deficient Cells 3575 MUTANT CELLS FRACTION FIG. 3. The effect of a-glucosidase inhibitors on oligosaccharide processing in parent and mutant cells. The ConA- Sepharose elution profiles are shown for the [2-3H]mannose-labeled glycopeptides derived from parent or mutant cells pulse-labeled for 15 min and chased for 6 h in the presence of no inhibitor (control), 4 mm DNJ, or 2 mm bu-dnj as described under Experimental Procedures. Elution with 10 mm a-methylglucoside (a-meglc) and 100 mm a-methylmannoside (a-meman) was begun as indicated by the arrows, aliquots of each fraction were counted, and the following fractions pooled for peaks Z (1-6), ZZ (11-15), ZZZ (16-25), and ZV (26-33). TABLE I1 Summary of oligosaccharide distribution in glycopeptides from cells mown in the absence or Dresence of a-ducosidase inhibitors Oligosaccharide pecies 3.7 3.2 Parent cells Mutant cells Control +DNJ +bu-dnj Control +DNJ +bu-dnj mol % of oligosaccharide 46.3 5545.5 39.5 47.8 3.6 7.2 7.4 5.3 5.7 5.6 5.0 3.1 3.6 4.5 7.2 6.2 4.2 8.9 tr. - - - 11.6 tr. - - - 45.2 3.4 4.0 2.8 control mutant cells are absent in the presence of either inhibitor, suggesting that their formation in the control cells is due to the residual a-glucosidase I1 activity of the mutant which is then totally inhibited by DNJ and bu-dnj. The bu-dnj-inhibited cells show the unique presence of Gl~~Man~-~GlcNAc oligosaccharides which indicates that bu- DNJ inhibits a-glucosidase I as well as a-glucosidase 11. In contrast to the mutant cells, the parent cell distribution of non-glucosylated oligosaccharides shown at the top of the table is changed dramatically in the presence of DNJ or bu- DNJ, approaching the levels seen in the mutant cells. One significant change is that MangGlcNAc and Man8GlcNAc are present in only trace amounts in DNJ and absent in bu-dnjtreated cells. This observation plus the presence of Glc2Man7- gglcnac with DNJ and both Glc2Man7-9GlcNAc and Gl~~Man~-~GlcNAc with bu-dnj indicates that these inhibitors are effectively blocking a-glucosidase I1 and in the latter case also a-glucosidase I activity. The essential point brought out by the experiment is that when these cells are blocked at a-glucosidase I1 whether by a mutation or an inhibitor they will utilize a bypass route to process a significant proportion of their glucosylated oligosaccharides. Endo a-mannosidase Activity-Just such a bypass route is provided by the endo a-mannosidase recently described by Lubas and Spiro (5) in rat liver Golgi membranes. They showed that this enzyme activity can excise a Glcal-3 Man disaccharide from GlclMangGlcNAc to generate a specific MansGlcNAc structure. They further showed that the endo a-mannosidase activity excised a Glc2Man and GlcsMan from the substrates Glc2MangGlcNAc and Glc3MangGlcNAc, respectively, although at a reduced rate compared to splitting of GlclMangGlcNAc (6). We postulated that such an endo a- mannosidase activity could be responsible for the oligosaccharide processing in the mutant cells, and accordingly we assayed both parent and mutant cell membrane preparations for endo a-mannosidase activity. When the membranes were incubated with [2-3H]mannose-labeled GlclMangGlcNAc and the released disaccharide GlclMan separated by paper chromatography, the results shown in Fig. 4 were obtained. Both - 4.4 - - 1.2 - - - 1.0 - - - 1.0-17.6 8.6 11.0 11.2 6.1 6.1 9.3 3.6 12.3 13.6 19.3 20.1-4.7 - - - 2.4 - - 6.5 1.7-4.2 Species not detected. 5.0 6.2 15.9 3.4 4.2 9.9 with endo H and the released oligosaccharides analyzed as before. Table I1 presents the distribution of oligosaccharides found under each set of conditions expressed as mol % of the total oligosaccharides. The type and % distribution of oligosaccharides in the control (noninhibited) parent and mutant cells is quite comparable to that for the 6-h chase cells shown in Table I, as expected. For the mutant cells, the distribution of the non-glucosylated species shown in the top half of the table is nearly identical in both control, DNJ, and bu-dnjtreated cells. However, the types of glucosylated oligosaccharides seen are different in the presence of either DNJ or bu- DNJ. The modest levels of GlclMan7-gGlcNAc observed in 201 / / t 2o Y 1 I 1 Y I I, I 2 3 75 150 225 HOURS pg PROTEIN FIG. 4. Endo a-mannosidase activity in parent and mutant cell membranes. Endo a-mannosidase assays were performed with [2-3H]mannose-labeled GlclMan9GlcNAc as substrate as described under Experimental Procedures. Panel A and B, parent cell membranes were assayed for 1, 2, or 3 h (149 pg of protein) (A) or 2 h with varying amounts of membrane (B). Panels C and D, mutant cell membranes were assayed for 1, 2, or 3 h (160 pg of protein) (C) or 2 h with varying amounts of membrane (D).

ant 3576 Oligosaccharide Processing in a-glc II-deficient Cells parent and mutant cell membranes catalyzed the reaction, the extent of which was proportional to time of incubation and amount of membrane protein up to about 60% splitting of the substrate. In the same experiment, GlczMan9GlcNAc, GlczMansGlcNAc, and GlczMan7GlcNAc were tested as substrates and shown to be split to produce the trisaccharide GlczMan, but at a much lower rate as shown in Table 111. The endo a-mannosidase activity is approximately the same in parent and mutant cell membranes, and, like the rat liver Golgi enzyme (6), it has better activity toward GlclMang- GlcNAc than toward GlczMan7~9GlcNAc. Among the Glcz oligosaccharides the ones with fewer mannose residues are better substrates. Evidence That Endo a-mannosidase Provides a Bypass Route-If the endo a-mannosidase is indeed the bypass route used in the mutant cells, one would expect these cells to produce an amount of GlczMan (and possibly some GlclMan) proportional to the amount of non-glucosylated oligosaccharides formed. If the activity is present in the Golgi apparatus, the saccharide products should be formed in the lumen of the Golgi cisternae and pass through the secretory pathway into the medium. To explore this possibility we set up a pulsechase experiment in which the medium was collected as a source of secreted tri- and disaccharides. This required some modifications in the usual procedure; the amount of [2-3H] mannose was increased and the labeling period was 30 min to get more radioactivity incorporated, the chase was initiated after thorough washing of the cells to remove the labeled mannose, and the cells were resuspended in low glucose medium without cold mannose to minimize the monosaccharide concentration in the medium. At the end of the 3-h chase, the medium was worked-up as described under Experimental Procedures utilizing an incubation with hexokinase and ATP to convert free mannose and glucose to their phosphorylated derivatives that could be removed on a mixed bed ion-exchange resin. After an initial paper chromatographic separa- tion which removed the bulk of the remaining radioactive mannose, the samples were rechromatographed on paper as shown in Fig. 5. The secretions from the mutant cells contained a prominent peak of radioactivity migrating between the markers maltose (G2) and maltotriose (G3) and corresponding to the trisaccharide produced in the in vitro endo a- mannosidase assays, and a smaller peak of radioactivity migrating faster than maltose with a mobility like the disaccharide produced in the in vitro endo a-mannosidase assays. A small amount of radioactive mannose was still present in the mutant secretions, but mannose represented essentially all of the radioactive saccharide found in the parent cell secretions which showed no trisaccharide or disaccharide peaks. The identity of the trisaccharide and disaccharide peaks from the mutant cell secretions as the expected glucosylated species was verified by enzymatic digestion with a-glucosidase and a-mannosidase as shown in Fig. 6. Panels A and D show that mock digestion of the tri- and disaccharide left their mobilities on paper chromatography unchanged; panels B and E show that neither was degraded by treatment with a- mannosidase indicating the absence of terminal nonreducing TABLE 111 Activity of endo a-mannosidase on various glucosylated substrates Parent Substrate % Split/lOO pg/h GlcsMangGlcNAc 0.6 0.6 GlcsManaGlcNAc 1.2 0.8 Glc,Man7GlcNAc 1.3 2.7 GlclMangGlcNAc 22.2 20.5 MUTANT IO 20 30 IO 20 30 CM FIG. 5. Paper chromatographic separation of the trisaccharide and disaccharide secreted by the mutant cells. Parent and mutant cells were pulse labeled with [2-3H]mannose for 30 min, washed, and chased for 3 h. The saccharides isolated from the chase media as described under Experimental Procedures, were separated by paper chromatography in solvent B, and aliquots of each eluted 1- cm strip were counted for radioactivity. Mobilities of standards run in an adjacent lane are shown by arrows: maltotriose (G3), maltose (G), and free mannose (M). TRISACCHARIDE 1501-.. 1501 DISACCHARIDE 50 liu l o 0 u IO 20 30 CM 10 20 30 I FIG. 6. Characterization of the secreted trisaccharide as GlczMan and the disaccharide as GlclMan. The centers of the trisaccharide and disaccharide peak from the mutant cell secretions (shown in Fig. 5) were each pooled, and aliquots of each were treated as follows prior to paper chromatography in solvent B. Panels A and D, mock digestion; panels B and E, digestion with a-mannosidase; panels C and F, digestion with a-glucosidase. One-cm strips of the paper were cut and counted for radioactivity. Mobilities of the standards are shown by the arrows: maltotriose (G3), maltose (Gz), and mannose (M). a-mannose residues; but panels C and F show that the radioactivity in both the trisaccharide and disaccharide was largely converted to free mannose upon digestion with yeast a-glucosidase. The residual unchanged tri- and disaccharide could represent contaminating non-glucosylated saccharides but more likely represent incomplete a-glucosidase action due in part to the very low substrate concentration in these reaction mixtures. We conclude that the trisaccharide is GlcpMan and the disaccharide is GlclMan. In order to quantitate the amount of secreted tri- and disaccharide formed and compare it to the amount of glycoprotein-bound oligosaccharide that had been processed in these cells, the cellular glycopeptides were prepared, fractionated on ConA-Sepharose, and their oligosaccharides analyzed as described for the other pulse-chase experiments. The results are shown in Table IV, and the distribution of various oligosaccharide species in the cellular glycoprotein is quite comparable to that seen after 3 h of chase in the experiment in Table I. To calculate the equivalence between mol %

Oligosaccharide Processing in a-glc 11-deficient Cells 3577 TABLE IV Distribution of oligosaccharides in mutant cell glycoproteins, secretions, and cell water Oligosaccharide species Mol % Total cpm in complex cpm/complex cpm/mol % 38.0 \ 180,000 51,500 1,350 0 0 50.5 IT 2.9 1.3 1.1 11.5 12.2 20.5 B. Secreted oligosaccharides GM 29,600 cpm GIM 24,300 cpm 18 mol % C. Oligosaccharides in cell water GzM 7,300 cpm 5.4 % } 10.4 mol % G?M 6.800 cdm 5.0 mol % oligosaccharide and radioactivity, the total cpm contained in fractions I and I1 from the ConA-Sepharose column (180,000 cpm), which constitute the complex oligosaccharide fraction, were divided by 3.5 (the number of radioactive sugar residues/ oligosaccharide) to determine the cpm/complex oligosaccharide (51,500). Since the complex oligosaccharides constitute 38 mol % of all the oligosaccharides then there are 1,350 cpm/ mol % oligosaccharides which can be used to calculate that the secretions contain 22 mol % trisaccharide and 18 mol % disaccharide for a total of 40 mol % of both, as shown in Table IV. During the experiment 50.5 mol % of non-glucosylated oligosaccharides were formed, and the saccharides recovered in the secretions can account for 80% of that processing. It occurred to us that perhaps some split saccharides had not yet exited the cells by the end of the experiment and were still in the lumen of the secretory system. In the workup of the cell pellet, the denatured residue left from the chloroform/methanol extraction is then extracted with water to remove water-soluble radioactivity prior to the final extraction with chloroform/methanol/water, and so we decided to look at that water extract as the fraction likely to contain intracellular split saccharides. After Sephadex G-25 separation of smaller molecular weight radioactivity, desalting on a mixed bed ion-exchange resin, and an initial paper chromatographic separation, the second paper chromatography of the water extract yielded a pattern like that seen in Fig. 5 for the secreted saccharides. As shown in Table IV, the water fraction contained 5.4 mol % GlczMan and 5 mol % GlclMan which account for the other 20% of the non-glucosylated oligosaccharides formed during the experiment. This experiment shows unequivocally that the endo a-mannosidase reaction is providing the bypass route for complex oligosaccharide synthesis in the a-glucosidase 11-deficient mutant cells. One remarkable result of this experiment was that the disaccharide GlclMan constituted such a large proportion of the split products, namely 23 mol % or 45.6% of all the saccharides formed, and presumably it was formed from GlclMan7_gGlcNAc intermediates whose steady-state level is low compared with the Gl~~Man~-~GlcNAc intermediates. The in vitro endo a-mannosidase assays also showed that GlclMangGlcNAc is split much better than GlczMan9GlcNAc, and this experiment indicates that in vivo the enzyme also preferentially attacks protein-bound monoglucosylated oligosaccharides. What was unexpected was that these a-glucosidase 11-deficient cells were generating so much Glcl Man7-gGlcNAc intermediate to serve as substrate for the endo a-mannosidase. However, a simple calculation reveals that this flux through Gl~~Man~~~GlcNAc intermediates is very low compared with the flux through monoglucosylated oligosaccharides in parent cells. In this experiment 23 mol % GlclMan plus the 5.3 mol % GlclMan7-9GlcNAc equals 28.5 mol % that had passed through the Glcl pool in 3 h or 9.5 mol %/h. The experiment with the parent cells in Table I shows that at the end of the 15-min pulse label, 56.1 mol % of non-glucosylated oligosaccharides plus 41.1 mol % GlclMangGlcNAc had already been formed for a total of 97.2 mol % flux through the Glcl pool in 15 min or 389 mol %/h. Thus, the rate of formation of GlclMan7_9GlcNAc species in mutant cells is only 2.5% their rate of formation in parent cells. It is noteworthy that when assayed for a-glucosidase I1 activity using 4-methylumbelliferyl a-glucoside as substrate, mutant cell extracts showed 1.3-2% of the activity of parent cell extracts (data not shown). This agrees with the value of 3% reported by Reitman et al. (3) using p-nitrophenyl a- glucoside as substrate. They also assayed the activity using ([3H]G1~)1Man7-9Gl~NA~ as substrate, and found that the mutant had less than 0.3% of parent activity. The assays with artificial a-glucoside substrates may be measuring some slight contribution by other a-glucosidases or the mutant a-glucosidase I1 may be altered in such a way that it can react with these artificial a-glucosides, and perhaps the terminal residue in Glcal-3GlcMan7~9GlcNAc, better than it can react with the terminal residue in Gl~al-3Man~_~GlcNAc. The kinetics of processing in the mutant cells as well as the processing intermediates found suggest that the PHAR2.7 mutant cells use the oligosaccharide processing pathway shown in Fig. 7. In the ER a-glucosidase I acts rapidly to convert Glc3Man9GlcNAc2 to GlczMangGlcNAcz, but the deficiency of a-glucosidase I1 allows only a small amount of GlclMangGlcNAc2 to be formed. The latter two oligosaccharides also appear to be converted to Glc2ManflGlcNAc2 and Glc,ManflGlcNAc2 in the ER by one of the ER a-man-

3578 Oligosaccharide Processing in cr-glc 11-deficient Cells Glc,Man,GlcNAc,cz-", GlczMongGlcNAcz--~Glc, Mon,GlcN&, 1 t ER FIG. 7. Oligosaccharide processing in a-glucosidase 11-deficient mutant cells. The pathway shown is catalyzed by the following enzymes: a-glucosidase I (I ); a-glucosidase I1 (2); ER a-mannosidase (3); Golgi a-mannosidase I (4); endo a-mannosidase (5). and the amount of split saccharides formed was equivalent to the amount of complex oligosaccharides formed. Their results in conjunction with our results in the current study show that when a-glucosidase I1 is blocked either by inhibitors or by lack of active enzyme as in the PHAR2.7 mutant cells, the endo a-mannosidase provides an alternate bypass route to the formation of complex oligosaccharide in substantial amounts. What is still unclear is what role the endo a-mannosidase plays in normal oligosaccharide processing in cells containing abundant a-glucosidase I1 activity. The fact that the normal parental cells did not secrete measurable amounts of di- and trisaccharide into the medium indicates that either they do not normally use this route of processing or they use it to such a small extent that the products escape detection in our procedure. Perhaps a small, but select, subset of glycoproteins are normally processed along the endo a-mannosidase route. Alternatively, this pathway may truly represent a bypass route the cells fall-back on when a-glucosidase I1 activity becomes limiting. nosidases before reaching the Golgi apparatus. If REFERENCES Glcl-,Man,G1cNAc, were to enter the Golgi and encounter the endo a-mannosidase, one would expect to have seen 1. Kornfeld, R., and Kornfeld, S. (1985) Annu. Reu. Biochem. 54, MansGlcNAcz among the oligosaccharides of the mutant 631-664 2. Hirshberg, C. B., and Snider, M. D. (1987) Annu. Reu. Biochem. cell glycoproteins, but it was conspicious by its absence. 56,63-87 The largest non-glucosylated oligosaccharide detected in 3. Reitman, M. L., Trowbridge, I. S., and Kornfeld, S. (1982) J. our analysis was Man7GlcNAcz which suggests that Biol. Chem. 257, 10357-10363 Gl~~-~Man~GlcNAc~ was the largest substrate acted on by the 4. Romero, P. A., and Herscovics, A. (1986) J. Biol. Chem. 261, endo a-mannosidase. There was no evidence from these ex- 15936-15940 5. Lubas, W. periments to support the other bypass mechanism, namely A., and Spiro, R. G. (1987) J. Biol. Chem. 262,3775-3781 transfer of non-glucosylated lipid-linked oligosaccharide di- 6. Lubas, W. A., and Spiro, R. G. (1988) J. Bid. Chem. 263,3990- rectly to protein. If that were occurring one would expect to 3998 see non-glucosylated high mannose oligosaccharides on the 7. Li, Y. T., and Li, S. C. (1972) Methods Enzymol. 288, 702-713 mutant cell glycopeptides immediately after the pulse label- 8. Trowbridge, I. S., Hyman, R., Ferson, T., and Mazaukas, C. ing. As shown in Table I, these were not seen, but one cannot (1978) Eur. J. Immunol. 8, 716-723 rule out the possibility that such transfers occur to a small 9. Mellis, S. J., and Baenziger, J. U. (1981) Ann. Biochem. 114, 276-280 extent and escape detection. 10. Bischoff, J., Liscum, L., and Kornfeld, R. (1986) J. Biol. Chem. While this manuscript was in preparation a very interesting 261,4768-4774 paper by Moore and Spiro (14) appeared in which the authors 11. Trevelyan, W. F., Proctor, D. P., and Harrison, J. S. (1950) report that HepG2 cells grown in the presence of DNJ utilize Nature 166,444-445 the endo a-mannosidase pathway to form complex oligosac- 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 charides and secrete GIMan, G2Man, and GBMan into the 13. Saunier, B., Kilker, R. D., Jr., Tkacz, J. S., Quaroni, A., and medium. In the presence of the glucosidase I inhibitor castan- Herscovics, A. (1982) J. Biol. Chem. 257, 14155-14161 ospermine, the cells produced mostly G3Man, but also GzMan 14. Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, and GIMan, indicating that glucosidase I1 was also inhibited, 13104-13112